This invention relates to isolated proteins and to peptides which are found on the surface of colon cells and colon cancer cells, as well as to nucleic acid molecules encoding said protein and peptides. The protein and peptides bind to tumor associated antibodies, such as mAb 33. The monomeric protein has a molecular weight of about 43 kD as determined by SDS gel electrophoresis under non-reducing conditions. In addition, this invention relates to the use of said nucleic acid molecules, protein, in monomeric or multimeric form, and to antibodies to said peptides in diagnostic, screening and therapeutic methods. This invention further relates to antibodies specific for said protein, in monomeric or multimeric form, and to antibodies to said peptides.
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1. Isolated nucleic acid molecule which encodes the protein portion of a protein containing molecule which binds to monoclonal antibody A33, wherein the protein portion has a molecular weight of from 40-45 kD as determined by SDS-PAGE under non-reducing conditions.
2. The isolated nucleic acid molecule of
3. The isolated nucleic acid molecule of
4. Expression vector comprising the isolated nucleic acid molecule of
6. A host cell transformed or transfected with the expression vector of
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This application is a continuation-in-part or Ser. No. 08/597,495, filed Feb. 2, 1996, now U.S. Pat. No. 5,712,369 which is a continuation-in-part of U.S. patent application Ser. No. 08/511,876 filed on Aug. 4, 1995, now abandoned entitled Colon Cell and Colon Cancer Cell Associated Protein and Peptides, both of which are incorporated herein by reference.
This invention is directed to human colon cell and colon cancer cell associated antigens, nucleic acid molecules, proteins and peptides. Specifically, the proteins and peptides of the invention, which are encoded by the nucleic acid molecules of the invention, are found both in and on the surface of human colon cells and human colon cancer cells, and bind to colon cancer antibodies. The protein, in a monomeric form, has a molecular weight of about 40-45 kD as determined by SDS gel electrophoresis under non-reducing conditions and about 49-55 kD when subjected to SDS-PAGE under reducing conditions. This protein, peptide fragments thereof and multimeric thereof can be used to develop reagents and methods useful in the diagnosis and treatment of cancer.
Colorectal carcinoma is a malignant neoplastic disease. There is a high incidence of colorectal carcinoma in the Western world, particularly in the United States. Tumors of this type often metastasize through lymphatic and vascular channels. Many patients with colorectal carcinoma eventually die from this disease. In fact, it is estimated that 62,000 persons in the United States and 8000 persons in Australia die of colorectal carcinoma annually.
To date, systemic therapies and chemotherapies have been developed for the treatment of colorectal cancer. However, no therapies have exhibited sufficient anti-tumor activity to prolong the survival of colorectal carcinoma patients with metastatic disease, with any degree of reliability. As a result, a need still exists to develop methods and products for the successful treatment of colorectal carcinoma.
Monoclonal antibody A33 is a murine immunoglobulin that has undergone extensive preclinical analysis and localization studies in patients (see Welt et al., J. Clin. Oncol., 8:1894-1906 (1990), Welt et al., J. Clin. Oncol., 12:1561-1571 (1994), and Welt et al. J. Clin. Oncol. 14: 1787-1797 (1996). This antibody binds to an antigen found in and on the surface of normal colon cells and colon cancer cells. This antigen is known as the A33 antigen.
In carcinomas originating from the colonic mucosa, the A33 antigen is expressed homogeneously in more than 95% of cases. The A33 antigen has not been detected in a wide range of other normal tissues studied. Its restricted expression defines this system as essentially "organ-specific" (colon, rectum and small bowel).
Immunofluorescence experiments have revealed that mAb A33 is internalized into the macropinosomes of A33 antigen-positive cells in vitro (Daghighian et al;. J. Nuc. Med., 37: 1052-1057 (1996). In a mouse model, mAb'A33 has been found to localize to xenografts of human colon cancer in substantial amounts, and it can be identified in the cytoplasm of transplanted colon cancer cells within the first hour after administration. Rapid tumor localization and high level of antibody uptake by tumors are thought to be related to the following factors: (1) A33 antigen is not secreted, and targeting of mAb A33 to tumor cells is therefore not impeded by shed A33 antigen diffusing from tumor cells to the vascular system; (2) mAb A33 is rapidly internalized into the cell once it binds to A33 antigen on the cell membrane, thereby increasing the amount of cell associated antibody; and (3) some colon cancer cell lines express large amounts of A33 antigen, binding up to 800,000 mAb A33 molecules per cell. Due to these properties, a need exists to isolate, characterize and sequence the A33 antigen, as well as related proteins with similar characteristics.
Many purification protocols typically utilize reduction steps in order to analyze proteins of interest by SDS-gel electrophoresis. In this way, proteins can be identified and monitored more easily. The inventors of the instant application found that surprisingly, by utilizing reducing conditions, they were unable to identify the target A33 protein by Western blotting. Standard techniques had to be changed so as to completely remove reducing steps in order to identify, monitor and characterize the A33 antigen of the invention. Once the antigen was isolated, studies on its behavior under reducing conditions could be carried out.
Purification of the A33 antigen has been further complicated by co-migration of other proteins, including actin, to about the same position on one and two dimensional gel electrophoresis. In addition, mAb A33 binds non-specifically to actin. The inventors of the instant application identified the Fc. regipon of the antibody as being responsible for the non-specific binding to actin. Removal of the Fc region has allowed the inventors to prevent actin binding. As actin is not a cell surface antigen on colon carcinoma cells, and is not sensitive to reduction, it became clear to the inventors that actin could not be the target for monoclonal antibody A33.
The difficulty in identifying, isolating and characterizing this antigen is evidenced by the fact that although the existence of the A33 antigen has been known for more than a decade, this is the first successful purification, isolation and sequencing of the antigen.
As described herein, the inventors of the instant application have identified, isolated and characterized the A33 antigen. The inventors have also isolated cDNA encoding the A33 antigen, determined the nucleotide sequence of the cDNA, and deduced the amino acid sequence for the A33 antigen. The A33 antigen, also referred to herein as the A33 protein, can be utilized to develop clinical reagents and methods useful in the prognosis, diagnosis and treatment of cancer and other diseases, in particular, cancers such as colon, rectum, gastric and small bowel mucosa cancer.
This invention is directed to an isolated protein which is found inside and on the surface of normal human colon cells and human colon cancer cells, as well as to peptide fragments of said protein. The protein and peptides are bound by the A33 colon cancer antibody or by polyclonal antibodies raised against regions of the protein sequence. When analyzed by SDS gel electrophoresis, the isolated glycoprotein of the invention has a molecular weight of about 40-45 kD, when non-reducing conditions are utilized and about 49-55 kilodaltons under reducing conditions. This invention further relates to nucleic acid molecules encoding said protein, and to the use of said glycoprotein, peptides and nucleic acid molecules in the diagnosis and treatment of cancer.
The above brief description, as well as further objects and features of the present invention, will be more fully understood by reference to the following detailed description of the presently preferred, albeit illustrative, embodiments of the present invention when taken in conjunction with the accompanying drawings wherein:
FIG. 1 represents cytofluorographic analysis of the LIM1215 and Hep-2 cells with A33 monoclonal antibody;
FIG. 2 shows that A33 antigen is detectable by Western blot after SDS gel electrophoresis using non-reducing conditions, but not detectable after SDS gel electrophoresis using reducing conditions. "-B-ME" indicates non-reducing conditions and "+-BME" refers to reducing conditions;
FIG. 3 shows immunoprecipitation of cell lysates with or without mAb A33;
FIG. 4 shows immunoprecipitation of cell lysates which were or were not incubated with tunicamycin;
FIG. 5 is comprised of FIGS. 5A and 5B, and represents Western blot analysis of A33 antigen extracted from LIM1215 cells under non-reducing conditions;
FIG. 6 shows biosensor analysis of the interaction between actin and either A33 IgG or the A33 F(ab)'2 fragment;
FIG. 7 is a flow chart depicting the chromatographic purification protocol used to purify A33 antigen;
FIG. 8 is comprised of FIGS. 8A and 8B and shows Western blot analysis of Triton X-100 and Triton X-114 extracts of LIM1215-colonic cells, respectively;
FIG. 9 shows anion-exchange HPLC of the A33 antigen;
FIG. 10 shows size-exclusion HPLC of the A33 antigen;
FIG. 11 is comprised of FIGS. 11A and 11B. FIG. 11A shows micropreparative RP-HPLC purification of Superose 12 active fractions. FIG. 11B shows biosensor analysis of the A33 antigen activity in the HPLC fraction;
FIG. 12 represents amino acid sequences of peptide fragments in the A33 antigen;
FIG. 13 is a flow chart which shows a protocol used for affinity purification of A33 antigen;
FIG. 14 represents Western blot analysis of sera obtained from mice, and rabbits immunized with chemically synthesized peptide SVETPQDVLRASQGKSVTLP (amino acids 2-21 of SEQ ID NO: 1) conjugated to keyhole limpet hemocyanin (KLH);
FIG. 15 represents Western blot analysis of the A33 antigen under non-reduced (panel 1) and reduced (panel 2) conditions using an anti-peptide IgG raised against the N-terminus of the A33 antigen;
FIG. 16 is comprised of FIGS. 16A and 16B. FIG. 16A, and its continuation FIG. 16B, represent the 2.6kb cDNA which encodes the A33 antigen;
FIG. 17 is a comparison of deduced amino acid sequences for human and murine A33;
FIG. 18 shows that mAb A33 precipitated labelled A33 antigen, following labelling with palmitate;
FIG. 19 depicts the abolition of staining when hydroxylamine was used.
Several colon cancer cultured cell lines, listed in Table 1, were obtained. The LIM1215 cell line was obtained from Ludwig Institute for Cancer Research, Melbourne, Australia. Cell lines SK-CO-17, SK-CO-19, SK-CO-10, SK-CO-11 and SK-CO-15 were obtained from Ludwig Institute for Cancer Research, New York, and Memorial Sloan Kettering Institute, New York. All other cell lines were obtained from the American Type Culture Collection, Rockville, Md.
Using the protocol described by Pfreundschuh et al., Proc. Natl. Acad. Sci. USA, 75:5122-5126 (1978), rosetting assays were performed on each of these cell lines using monoclonal antibody A33 (mAb A33), secreted by a hybridoma cell line which was deposited under the Budapest Treaty with the American Type Culture Collection, Rockville, Md. and catalogued as ATCC No. HB 8779. mAb A33 has an isotype of IgG2a and, as described herein, binds to an antigen denoted A33 which is present in and on the surface of human colon carcinomas. Several of the colon carcinoma cell lines were found to be A33-positive, as determined by resetting assays, immunoassays and immunohistochemistry (see Table 1 which follows).
| TABLE 1 |
| REACTIVITY OF mAb A33 WITH HUMAN COLON |
| CANCER CELL LINES |
| Rosetting A. Western |
| Cell Line Titer Blot Immune precip. |
| A33 Positive Colon Lines |
| LIM 1215 213 +++ ++ |
| LOVO 212 + |
| LS 174T 212 |
| LS 180 211 ++ |
| NCI-H508 212 +++ ++ |
| SK-CO-17 29 + |
| SK-CO-19 213 |
| SNC-2B 212 |
| SW403 213 + |
| SW1222 212 +++ ++ |
| COLO 205 + + |
| ASPC-1 (pancreatic) 213 ++ ++ |
| A33 Negative Colon Lines |
| DLD1 -- |
| HCT15 -- -- |
| HT29 -- -- |
| SK-CO-10 -- -- |
| SK-CO-11 -- |
| SK-CO-15 -- |
| SW480 -- |
| SW620 -- -- -- |
| SW837 -- |
| SW1116 -- -- |
| SW1417 -- |
The LIM1215 colonic cell line, which was positive in the resetting assays of Example 1, was grown in RPMI medium containing 10% fetal calf serum. Confluent cells (106 /cm2) were passaged using Trypsin-Versene solution. Cells were seeded 1/10 into tissue culture dishes containing 25 ml RPMI 1640 supplemented with 10% fetal calf serum, 1 μg/ml hydrocortisone, 0.025 U/ml insulin and 10.82 μg/ml α-thioglycerol. The dishes were incubated at 37°C in an atmosphere of 5% CO2 for 5 days. After removing the media, cells were washed with PBS before being removed from the surface using a cell scraper. Cells were washed in PBS and resuspended at 109 cells/ml.
A33 antigen expression on the surface of the LIM1215 colonic carcinoma cell line was then analyzed by flow cytometry following standard techniques. The Hep-2 epidermoid carcinoma cell line (Boring et al., Cancer J. Clin., Vol. 44, pp. 7-26 (1994)) was used as a negative control. The cells were washed and resuspended at 5×106 cells/ml in 500 μl of PBS containing 5 mM EDTA and 5% fetal calf serum. The cells were incubated with 5 μg A33 mAb for 30 minutes at 4°C After washing with buffer, the cell/antibody complexes were incubated with fluorescein-conjugated anti-murine IgG (1/50 dilution). A negative control was performed by staining the cells with an isotopically matched non-related antibody (5 μg) followed by fluorescein-conjugated anti-murine IgG alone. Flow cytometry was performed using a FACScan flow cytometer (Becton Dickinson, San Jose, Calif., U.S.A.).
FIG. 1 shows cytofluorographic analysis of LIM1215 and Hep-2 cells with A33 monoclonal antibody. The entire population of LIM1215 cells exhibited a strong homogeneous fluorescence (panel B) when incubated with A33 mAb, compared with the fluorescence obtained with the control antibody (panel A). The profiles shown in the panels obtained with the Hep-2 cells (C and D) were overlapping, indicating no detectable A33 mAb binding to these cells. The X axis shows the fluorescence intensity (log scale) and the Y axis shows the cell number.
Cell lines which were A33-positive in resetting assays (Table 1) were lysed using 0.3% Triton X-100 in PBS, pH 7.4. Other detergents known to those skilled in the art can also be used to lyse A33-positive cells. The cell lysates of nine A33-positive cell lines and, in addition, five A33-negative cell lines (controls) were probed for A33 antigen expression by Western blot analysis using non-reducing conditions. A molecule with a molecular weight of about 43 kD was detected by Western blotting with mAb A33 in lysates from colon cancer cells which were A33-positive by resetting assay. This molecule was not detected in lysates obtained from cell lines which tested negative for A33 in resetting assays, or by antibodies other than mAb A33, including anti-actin mAb. The A33 antigen was detectable by Western blot analysis only after SDS gel electrophoresis using non-reducing conditions. The A33 antigen was not detectable using reducing conditions. The Western blot shows in FIG. 2 utilized A33 antigen obtained by affinity purification from SW1222 cells. The upper band (FIG. 2) indicates multimeric form of the A33 protein.
A33 antigen was immunoprecipitated from colon carcinoma cell lysates. In order to do this, colon cancer cells were labeled with 3 H-GlcNAc or 35 S using standard techniques known to those skilled in the art. Cell lysates which were A33 positive by rosetting assays, and which exhibited a band of about 43 kD by SDS gel electrophoresis under non-reducing conditions, were immunoprecipitated with monoclonal antibody A33.
FIG. 3 shows that a molecule was immunoprecipitated from A33-positive lysates which has a molecular weight of about 43 kD. This band was not precipitated by lysates which were A33-negative in rosetting assays. In addition, this band was not precipitated by antibodies other than mAb A33 ("no. 1°Ab" indicates that mAb A33 was not used). Since 3 H-GlcNAc is a carbohydrate, which is incorporated into the glycosylation side of glycoproteins, these results suggest that the A33 antigen contained in the band is a glycoprotein. Additional evidence in support of this is provided in examples which follow.
35 S-labeled SW1222 cells were incubated with 5 μg/ml tunicamycin for 18 hours. Tunicamycin is known to block N-glycosylation of glycoproteins. These cells, as well as cells which were not incubated with tunicamycin, were lysed and subjected to immunoprecipitation with A33 antibody, FB-5 antibody (control) or no antibody (control). FIG. 4 shows the immunoprecipitation results.
Of the cells which were not incubated with tunicamycin, immunoprecipitation with A33 antibody showed a band at about 43 kD. Immunoprecipitation with antibody FB-5, which was an isotype control or no antibody, showed no such 43kD band. Of the cells incubated with tunicamycin, immunoprecipitation with A33 antibody showed a band at 43kD, as well as three other bands of lower molecular weight. These lower molecular weight bands indicate the presence of A33 antigen wish a different degree of glycosylation due to the presence of tunicamycin. This provides further evidence that the A33 antigen is a glycoprotein, and contains N-linked oligosaccharides.
A33 antigen was identified using 2-dimensional gel electrophoresis under non-reducing conditions. First, the LIM1215 colonic cell line was grown in RPMI medium containing 10% fetal calf serum. Confluent cells (106 /cm2) were detached from the plastic dish using Trypsin-Versene solution. Cells were seeded 1/10 into tissue culture dishes (150×20 mm) containing 25 ml RPMI 1640 supplemented with 10% fetal calf serum, 1 μg/ml hydrocortisone, 0.024 U/ml insulin and 10.82 μg/ml α-thioglycerol, as described above. Dishes were incubated at 37°C in an atmosphere of 5% CO2 for 5 days. After removing the media, cells were washed with PBS before being removed from the surface using a cell scraper. Cells were washed in PBS and resuspended at 109 cells/ml.
Next, A33 antigen was extracted from 3×108 LIM1215 cells using 0.3% Triton X-100 in 10 mM Tris-HCl buffer (pH 7.4).. The extract was diluted 1:1 with sample buffer comprised of arginine/lysine buffer, pH 10, containing 30k glycerol, and electrophoresed on small (8×8 cm) Novex 2-dimensional gel electrophoresis gels under non-reducing conditions.
The proteins were separated in the first dimension by isoelectric focusing at a pH of 3.5-8.5, and in the second dimension by SDS-PAGE (10% acrylamide gels). The A33 antigen was localized in the gel by staining with Coomassie Blue R-250, along with immunoblot analysis using mAb A33 (FIG. 5B). For comparison, the staining pattern observed using an anti-actin mAb (FIG. 5A) is shown. Actin is used for comparison because it has similar migration characteristic to the A33 antigen.
Biosensor analysis was performed on the LIM1215 cell extracts and chromatographic fractions. The extracts and fractions were monitored using ah' instrumental optical biosensor (BIAcore™, Pharmacia Biosensor, Uppsala, Sweden), with a F(ab)'2 fragment of A33 humanized monoclonal antibody immobilized onto the biosensor surface.
To prepare the F(ab)'2 fragment, A33 antibodies were purified (King et al.; Br. J. Cancer, Vol. 72, pp. 1364-1372 (1995)). F(ab)'2 fragments were generated by pepsin (1% w/w) digestion of 10 mg A33 mAb in 0.1 M sodium acetate (pH 3.5). These were then purified by size exclusion chromatography on a Sephacryl S-200 (2.8×60 cm) column (Pharmacia Biotech) equilibrated with 50 mM sodium phosphate (pH 7.4) containing 0.15 mM NaCl. The elution was performed at a flow rate of 0.5 ml/min.
The detection of the antigen binding to the F(ab)'2 fragment is based on the phenomenon of surface plasmon resonance, a technique which measures small changes in refractive index at, or near to, the gold sensor surface. Prior to the biosensor assay, cell extracts and chromatographic fractions were diluted to 100 μl, final volume in BIAcore™ buffer (HBS): 10 mM Hepes (pH 7.4) containing 3.4 mM EDTA, 0.15 mM NaCl and 0.005% Tween 20. Samples (30 μl) were injected over the sensor surface at a flow rate of 5 μl/min. Following completion of the injection phase, dissociation was monitored in BIAcore™ buffer at the same flow rate for 360 seconds. Residual bound antigen was eluted and the surface regenerated between injections using 40 μl of 10 mM NaOH. This treatment did not denature the protein immobilized onto the sensor surface as shown by equivalent signals on reinjection of a sample containing the A33 antigen.
FIG. 6 shows biosensor, analysis of the. interaction between actin and either the complete A33 antibody or the A33 F(ab)'2 fragment. A preparation of rabbit, muscle actin (0.3 μg) was injected at a flow rate of 5 μl/min over a sensor surface which had been immobilized/with either whole A33 (upper trace) or A33 F(ab)'2 fragment (lower trace). Protein/protein interactions were-monitored by surface plasmon resonance. At the end of the injection pulse, a signal of 247 RU was observed due to actin binding to A33 IgG, while the signal corresponding to actin binding to A33 F(ab)'2 was only 4 RU (as indicated by arrows).
A33 antigen was purified from LIM1215 cells for sequence analysis. FIG. 7 is a flow chart which depicts the chromatographic purification protocol used to purify A33 antigen. To extract A33 antigen, LIM1215 colonic cells (2×109 cells) were harvested, washed in phosphate-buffered saline (PBS) and solubilized (108 cells/ml) for 30 minutes at 4°C with either 0.3% (v/v) Triton X-100 or 1% (v/v) Triton X-114 in 15 mM Tris-HCl (pH 7.4) containing 1 mM PMSF, 1 mM pepstatin, 0.1 mM leupeptin and 0.01 U/ml aprotinin. The resulting extracts were centrifuged twice at 4°C for 20 minutes at 14,000g. The Triton-X100 supernatant was taken directly for Green-Sepharose HE-4BD chromatography. The Triton X-114 extracted supernatant was layered over 6% sucrose in 15 mM Tris-HCl (pH 7.4) with 0.06k (v/v) Triton X-114, containing the protease inhibitors listed above. The tubes containing the Triton X-114 extracts and the sucrose were incubated at 37°C for 30 minutes and then centrifuged at 25°C for 15 minutes at 5,000 g. The detergent phase was collected for chromatographic purification.
In order to perform Green-Sepharose chromatography, Triton-X100 extracts or the Triton X-114 detergent phase were diluted to a final concentration of 0.1% Triton and loaded at 4°C onto a Green-Sepharose HE-4BD column (100×10 mm ID) connected to a Fast Protein Liquid Chromatography system (FPLC, Pharmacia Biotech, Uppsala,, Sweden). The column was equilibrated with 10 mM Tris-HCl (pH 7.4) containing 0.1t CHAPS (w/v). Bound proteins, including actin, were eluted stepwise with 1M NaCl. The breakthrough contained the A33 antigen, and was collected for anion-exchange HPLC, as described below.
Western blot analysis was performed throughout purification to confirm the presence of A33 antigen. Electrophoresis and Western blot analysis were performed on precast Phastgels using a Phastsystem separation and control unit (Pharmacia Biotech). Cell extracts and chromatographic fractions were electrophoresed under non-reducing conditions as described by Reid et al, Electrophoresis, Vol. 16, pp. 1120-1130 (1995), on 8-25% SDS-PAGE Phastgels or 8-25% native Phastgels and transferred onto PVDF membranes and incubated with A33 monoclonal antibody. RP-HPLC purified A33 antigen was also analyzed by Western blot under non-reducing and reducing conditions using polyclonal anti-N-terminal peptide antibodies (described herein). IgG binding was probed with horseradish peroxidase-labelled goat anti-mouse IgG, goat anti-human IgG or goat anti-rabbit IgG and detected by enhanced chemiluminescence (ECL).
FIG. 8 shows Western blot analysis of the Triton X-100 and Triton X-114 extracts of LIM1215 colonic cells. Panel A shows the following: Lane 1: LIM1215 cells solubilized in 0.3% Triton X-100. Lane 2: Green-Sepharose breakthrough containing the 43K A33 antigen. Lane 3: Green-Sepharose binding proteins eluted with 1M NaCl containing a 41 kD molecular weight band. Lane 4: Rabbit muscle Actin (1 μg).
Panel B shows the following: Lane 1: LIM1215 cells solubilized in 1% Triton X-114. Lane 2: Txiton X-114 aqueous phase. Lane 3: Triton X-114 detergent phase. Lane 4: Green-Sepharose breakthrough. Lane. 5: Green-Sepharose binding proteins eluted with 1M NaCl.
Following Green-Sepharose chromatography (described above), anion-exchange HPLC was performed. The Green-Sepharose breakthrough was injected at 4°C onto a Mono Q HR 10/10 column previously equilibrated in 10 mM Tris-HCl (pH 7.4) containing 0.1% (w/v) CHAPS. The proteins were eluted from the column using a linear 0-1M NaCl gradient generated over 90 minutes at a flow rate of 1 ml/min. Fractions (1 ml) were collected automatically using a fraction collector (FRAC 100, Pharmacia Biotech). Proteins were detected by absorbance at 280 nm. The A33 antigen was detected using both Western blotting under non-reducing conditions and biosensor analysis.
FIG. 9 shows anion-exchange HPLC of the A33 antigen. The proteins contained in the Green-Sepharose breakthrough fraction which were loaded onto the Mono Q HR 10/10 anion-exchange column and eluted at a flow rate of 1 ml/min with a linear gradient from 0-1 M NaCl are indicated (--). One ml fractions were collected and aliquots (20 μl) of each of the fractions were taken for biosensor assay. The approximately 43 kD antigen was detected in the labelled fractions by Western blot analysis under non-reducing conditions (inset, FIG. 9) as described herein.
Next, size-exclusion HPLC was performed. The active fractions eluted from the Mono Q column (10 ml) were concentrated 10 fold using a Speed Vac concentrator (Savant Instruments Inc., N.Y., U.S.A.), dialyzed against PBS containing 0.05% CHAPS (w/v) and loaded at 4°C onto a Superose 12 HR 10/30 column. Proteins were eluted with PBS containing 0.05% (w/v) CHAPS at a flow rate of 500 μl/min. Fractions (0.5 ml) were collected. Proteins were detected at 280 nm and the A33 antigen was monitored using both Western blotting and biosensor analysis as described above.
FIG. 10 shows size-exclusion HPbC of the A33 antigen. The elution positions of protein calibration standards (BSA dimer, BSA and trypsin inhibitor) are indicated above the chromatographic trace. The A33 antigen was also detected by Western blot analysis under non-reducing conditions (inset A) in the fractions indicated. Immunoblot analysis of a pool of the Superose 12 activity (fractions 2-5) using an 8-25% native gel revealed that the A33 antigen migrated under native conditions (no SDS) with a relative molecular mass of 180 kD (inset B, FIG. 10).
Reversed-phase HPLC chromatography was then performed. Superose 12 active fractions (2.5 ml) were loaded at a flow rate of 1 ml/min, by multiple 1 ml injections, onto a Brownlee Aquapore RP 300 micropreparative RP-HPLC column (30×2.1 mm ID) equilibrated with the primary solvent, 0.15% (v/v) trifluoroacetic acid (TFA) in water. The proteins were eluted with a linear 60 minute gradient to 60% aqueous n-propanol/0.125% (v/v) TFA at a flow rate of 100 μl/min. The column temperature was 45°C Protein detection was performed at 215 nm. The A33 antigen was detected: using both Western blotting and biosensor analysis. The peak containing the A33 antigen was repurified and further concentrated using a Brownlee Aquapore RP 300 micropreparative RP-HPLC column (100×1 mm ID) prior to N-terminal sequence analysis, using the gradient conditions described above at a flow rate of 50 μl/min. Eluent fractions were recovered manually.
FIG. 11 shows micropreparative RP-HPLC purification of Superose 12 active fractions. Panel A, main frame, shows the elution profile of the fractions from micropreparative RP-HPLC as analyzed by absorbance at 215 nm and by biosensor. Panel A, inset A, shows aliquots (2 μl) of each fraction, analyzed by SDS-PAGE (8-25% gel, silver stained), and Panel A, inset B, shows a Western blot under non-reducing conditions. Panel B shows biosensor analysis of individual fractions from micropreparative RP-PHLC. Aliquots (20 μl) of each fraction were concentrated using a Speed Vac concentrator and redissolved in 100 μl of RIAcore™ buffer. 30 μl aliquots were analyzed using the biosensor. Activity was found in the fractions eluting between 46 and 48 minutes.
As discussed above, the A33 antigen-containing reversed-phase HPLC fractions were pooled for amino acid sequence analysis. N-terminal amino acid sequence analysis of purified A33 antigen/protein was performed on a Hewlett-Packard model G1005A protein sensor operated with the routine 3.0 sequencer program described by Reid et al., Electrophoresis, Vol. 16, pp. 1120-1130 (1995). The following N-terminus sequence of 30 amino acids was obtained: (SEQ ID NO: 1)
XSVETPQDVLRASQGKSVTLPXTYHTSXXXREGLIQWD.
A search of all of the available protein, DNA and expressed sequence tag databases did not reveal any significant amino acid sequence identity of the A33 N-terminus with known proteins.
In addition, A33 antigen-containing reversed phase HPLC fractions were subjected to tryptic digestion as described by Simpson et al., Eur. J. Biochem., Vol. 183, pp. 715-722 (1989). Peptide fragments T1 and T2 were obtained. The amino acid sequences for these peptide fragments are shown in FIG. 12.
A33 antigen-containing fractions were obtained from SW1222 cells utilizing the protocol shown in FIG. 13. To perform affinity chromatography, the A33 affinity column was prepared according to the protocol described by Schneider et al., J. Biol. Chem. Vol. 257, pp.. 10766-10769 (1982). A33 monoclonal antibody was diluted to 1 mg/ml in 0.1 M borate, pH 8.2, and incubated overnight at 4°C with 1.5 ml Protein A-Sepharose. After washing with 0.1 M borate, pH 9.2, the Protein-A-monoclonal/antibody complex was incubated for 1 hour at room temperature with 20 mM dimethylpimelimidate in 0.1 M borate, pH 9.2. Non-covalently bound antibody was removed with 50 mM glycine, pH 2.5. The remaining active dimethylpimelimidate groups were deactivated by washing and incubating the beads with 0.1 M ethanolamine pH 8∅
The reversed-phase HPLC fractions were pooled for amino acid sequence analysis sequence analysis was performed as described in Example 13. The following A33 N-terminus sequence was obtained: (SEQ ID NO: 4)
ISVETPQDVLRASQGKSVTLPXTYHTSTSSREGLIQWDKL
A sequence search did not reveal any significant amino acid sequence identity with known proteins. This N-terminus sequence was utilized to obtain the cDNA sequence which encodes A33 antigen (described below).
In addition, A33 antigen-containing reversed phase HPLC fractions were subjected to Asp-N endoproteinase digestion as described by Simpson et al., Eur. J. Biochem., Vol. 183, pp. 715-722 (1989). Peptide fragments D1, D2, D3 and D4 were obtained. These peptides were purified by micropreparative RP-HPLC. The amino acid sequences for these peptide fragments are shown in FIG. 12. It was determined that there was a lack of an amino acid in cycle 3 of the Edman degradation of peptide D4. Asp 112 was flanked by Thr at position 114. As this is a classical N-glycosylation motif, evidence was provided that A33 protein is N-glycosylated.
Fractions were also subjected to pepsin digestion, as described by Sarkar et al, Proc. Nat'l Acad. Sci. U.S.A., Vol. 88, pp. 234-238 (1991). Peptide fragment P1 was obtained. The amino acid sequence for peptide fragment P1 is shown in FIG. 12.
RP-HPLC fragments were subjected to Thermolysin/pepsin/Asp-N digestion. Thermolysin digestion was performed as described by Sarkar, supra. Peptide fragments Pc1 and Pc2 were obtained. The amino acid sequences for peptide fragments Pc1 and Pc2 are shown in FIG. 12.
Immunization studies were performed utilizing an immunogen derived from the amino acid sequence of the N-terminus of A33 antigen. A chemically synthesized peptide, SVETPQDVLRASQGKSVTLP (amino acids 2-21 of SEQ ID NO: 1) was conjugated to KLH and injected,, with adjuvant, into two mice and into two-rabbits. Rabbits were immunized four times at three week intervals. In the first immunization, complete Freund's adjuvant (CFA) was used. In subsequent rabbit immunizations, incomplete Freund's adjuvant (IFA) was used. Mice were immunized four times at two week intervals, using standard adjuvant. Sera were obtained from the rabbits and from the mice. The sera were subjected to Western blot analysis.
It was found that both the rabbits and mice developed IgG antibodies which reacted with the peptide, and also with the 43 kD band (the same 43 kD band was recognized by mAb A33) in using cell line SW1222 (FIG. 14). IgG was purified from rabbit immune sera by Protein-A affinity chromatography. Purified IgG was characterized by SDS-PAGE and Western blot analysis for reactivity with LIM1215 cell lysates and purified A33 antigen. The IgG was found to react strongly with the 20 amino acid peptide discussed supra, and with the approximately 43 kD protein which was recognized by mAb A33 under non-reducing conditions. In addition, rabbit IgG anti-serum reacted strongly with whole A33 antigen in reduced form (FIG. 15). HPLC purified A33 antigen from LIM1215 (0.1 μg) was electrophoresed on an 8-25% SDS-PAGE Phastgel under non-reducing (FIG. 15, lane 1) and reducing (FIG. 15, lane 2) conditions and analyzed by Western blot using an anti-peptide IgG raised against residues 2-21 of SEQ ID NO: 1, as described-above. The A33 antigen N-terminus sequence, and fragments thereof, can be used to develop A33 antigen-specific antibodies. These antibodies will recognize and bind, to A33 antigen or fragments thereof, in either reduced or non-reduced form.
The amino acid sequence of the A33 N-terminus of A33 protein was used to clone A33 protein cDNA. Poly (A)+ RNA (80 μg) was prepared in-house from confluent LIM1215 cells by two rounds of enrichment on columns of oligo (dT) cellulose using standard procedures. A LIM1215 cDNA library was custom-synthesized in the λZAPII expression vector by Clontech (Palo Alto, Calif., U.S.A.) using oligo (dT) and random hexamer primers to prime first strand DNA synthesis (standard procedures), using this mRNA.
Successful screening of the library was achieved with a DNA probe generated from the LIM1215 cDNA library using the polymerase chain reaction (PCR). Six 17mer antisense oligonucleotides (R9-R14), each with only 8-fold degeneracy, were designed to correspond to amino acid residues 34-39 of the A33 antigen N-terminal sequence (L I Q W D K (amino acids 34-39 of SEQ ID NO: 4)) as follows:
TBL Primer #1477 (R9) 5' A(R) (Y) TT (R) TCCCACTGAAT (SEQ ID NO: 12) Primer #1478 (R10) 5' A(R) (Y) TT (R) TCCCATTGAAT (SEQ ID NO: 13) Primer #1479 (R11) 5' A(R) (Y) TT (R) TCCCACTGGAT (SEQ ID NO: 14) Primer #1480 (R12) 5' A(R) (Y) TT (R) TCCCATTGGAT (SEQ ID NO: 15) Primer #5915 (R13) 5' A(R) (Y) TT (R) TCCCACTGTAT (SEQ ID NO: 16) Primer #5916 (R14) 5' A(R) (Y) TT (R) TCCCATTGTAT (SEQ ID NO: 17)These were paired with sense primers designed to hybridize to sequences present in the backbone of the λZAPII vector and were used in PCR reactions with the amplified LIM1215 cDNA library as the source of A33 antigen template. The successful reaction occurred with primers described below. For PCR reaction, the template used was amplified LIM1215 cDNA library in λZAPII vector. The primers used were as follows: KS primer 5'CGAGGTCGACGGTATCG (SEQ ID NO: 18) (l7mer) (hybridizes to a sequence in multicloning site of λZAPII vector); and R10 primer (described above).
The reaction conditions were as follows:
TBL cDNA library (10" pfu/ml) 1 μl 10 × T'aq ™ buffer 5 μl 1.5 mM NTPs 4 μl 15 mM MgCl2 5 μl KS (50 pmoles/μl) 1 μl R10 (50 pmoles/μl) 1 μl Water 32.5 μl Taq polymerase 0.5 μl (added last in Hot 50.0 μl Start)The touchdown program used in the PCR was as follows:
TBL 1 95°C × 5 min 2 95°C × 1 min 3 60°C × 1 min -2°C in subsequent cycles 4 72°C × 2 min 5 Go to (2) eleven times 6 95°C × 1 min 7 37°C × 2 min 8 72°C × 2 min 9 95°C × 1 min 10 45°C × 2 min 11 72°C × 2 min 12 Go to (9) thirteen times 13 72°C × 5 min 14 4°C holdThree products were generated, and were 1.4 kb, 0.5 kb, and 0.3 kb long.
The 1.4 kb product (designated R10/1) and the 0.5 kb product (designated R10/2) were separated on a 3% agarose gel and purified using the Bresa-clean™ nucleic acid purification kit (Bresatec, Adelaide, S. Australia). These purified products were used as templates in further PCR reactions in order to generate a greater yield of product. PCR reactions were conducted exactly as described supra, except that 1 μl of purified PCR product (either RIO/1 or R10/2) was used as DNA template instead of 1 μl of the LIM1215 cDNA library.
The R10/1 PCR reaction produced two bands:
TBL Upper band Size 1.4 kb (very faint) Lower band Size 0.3 kb (strong) designated 10/1 300 bpThe R10/2 PCR reaction produced two bands:
TBL Upper band Size 0.5 kb (strong) Lower band Size 0.3 kb (strong) designated 10/2The 0.3 kb fragments (10/1 300bp and 10/2) were gel-purified as described above. Nucleotide sequencing of both fragments was conducted and the reverse complement of each sequence was found to encode a portion of the A33 N-terminal protein sequence.
The following precise primers to the A33 antigen cDNA sequence were then synthesized in order to amplify a precise 189 bp PCR product for use as a probe to screen the LIM1215 cDNA library.
Primer #1747 (A33wsense primer 1) 5'CCTGTCTGGAGGCTGCCAGT (20mer) (SEQ ID NO: 19)
Primer #1748 (A33 antisense primer 1) 5'AGGTGCAGGGCAGGGTGACA (20mer) (SEQ ID NO: 20)
The above primers were used in a standard PCR reaction as follows, and generated a product of the predicted size (189 bp).
Standard PCR Reaction Conditions
TBL 10/1-300 bp product 1 μl 10 × T'aq buffer 2 μl 2.5 mM NTPs 1.6 μl 15 mM MgCl2 2 μl Primer #1747 (50 pmoles/μl) 1 μl Primer #1748 (50 pmoles/μl) 1 μl Water 11 μl Taq polymerase 0.4 μl (added last) 20.0 μlStandard PCR program as follows:
TBL 1 95°C × 5 min 2 95°C × 1 min 3 55°C × 1 min 4 72°C × 1 min 5 Go to (2) thirty times 6 72°C × 5 min 7 4°C holdThe 189 bp product was separated on a 3% agarose gel and purified using the Bresa-clean™ kit. It was then radio-labelled with [α32 P]ATP and [α32 P]CTP to a specific activity of >107 dpm/μg DNA using well known random primer reaction and Klenow polymerase procedures and used to screen 800,000 clones of the LIM1215 cDNA library (standard procedures). After three rounds of screening thirteen purified A33 antigen cDNA clones were obtained, the longest of which was approximately 2.8 kb. See infra.
The labelled PCR probe was also used in Northern analysis and produced a strong hybridizing signal with a single species of mRNA of size approximately 2.8 kb in total RNA and poly (A)+ enriched RNA from LIM1215 cells, suggesting that the 2.8 kb clone was likely to be close to full-length. Several clones were sequenced and all were found to encode the A33 antigen N-terminal protein sequence. The complete nucleotide sequence of a 2.6 kb clone (clone 11) is depicted in FIG. 16.
When one 2.6 kb cDNA clone was radiolabelled as described above (i.e., using [α32 P]ATP and [α32 P]CTP in the random primers reaction with Klenow polymerase) and used in Northern analysis, a strong signal of size approximately 2.8 kb was obtained with total RNA prepared from A33 antigen positive cell lines (LIM1215, LIM1899 and LIM1863) and normal human colonic epithelial tissue, but not with total RNA form A33 antigen negative cell lines (LIM2099, LIM2405, LIM2537).
The 319 amino acid translation protein product (A33 antigen) was deduced from nucleotide sequence of several 2.6 kb clones. It was predicted that protein translation is initiated at the second ATG from the 5' end in the cDNA sequence. This was deduced by reference to the Kozak consensus sequence (GCCC(R)CCATGG (SEQ ID NO: 21)) for initiation of translation. The deduced full length translation protein product comprises 319 amino acids, and has the following amino acid sequence (SEQ ID NO: 22)
| Met Val Gly Lys Met Trp Pro Val Leu Trp Thr Leu Cys Ala Val Arg |
| 5 10 15 |
| Val Thr Val Asp Ala Ile Ser Val Glu Thr Pro Gln Asp Val Leu Arg |
| 20 25 30 |
| Ala Ser Gln Gly Lys Ser Val Thr Leu Pro Cys Thr Tyr His Thr Ser |
| 35 40 45 |
| Thr Ser Ser Arg Glu Gly Leu Ile Gln Trp Asp Lys Leu Leu Leu Thr |
| 50 55 60 |
| His Thr Glu Arg Val Val Ile Trp Pro Phe Ser Asn Lys Asn Tyr Ile |
| 65 70 75 80 |
| His Gly Glu Leu Tyr Lys Asn Arg Val Ser Ile Ser Asn Asn Ala Glu |
| 85 90 95 |
| Gln Ser Asp Ala Ser Ile Thr Ile Asp Gln Leu Thr Met Ala Asp Asn |
| 100 105 110 |
| Gly Thr Tyr Glu Cys Ser Val Ser Leu Met Ser Asp Leu Glu Gly Asn |
| 115 120 125 |
| Thr Lys Ser Arg Val Arg Leu Leu Val Leu Val Pro Pro Ser Lys Pro |
| 130 135 140 |
| Glu Cys Gly Ile Glu Gly Glu Thr Ile Ile Gly Asn Asn Ile Gln Leu |
| 145 150 155 160 |
| Thr Cys Gln Ser Lys Glu Gly Ser Pro Thr Pro Gln Tyr Ser Trp Lys |
| 165 170 175 |
| Arg Tyr Asn Ile Leu Asn Gln Glu Gln Pro Leu Ala Gln Pro Ala Ser |
| 180 185 190 |
| Gly Gln Pro Val Ser Leu Lys Asn Ile Ser Thr Asp Thr Ser Gly Tyr |
| 195 200 205 |
| Try Ile Cys Thr Ser Ser Asn Glu Glu Gly Thr Gln Phe Cys Asn Ile |
| 210 215 220 |
| Thr Val Ala Val Arg Ser Pro Ser Met Asn Val Ala Leu Tyr Val Gly |
| 225 230 235 240 |
| Ile Ala Val Gly Val Val Ala Ala Leu Ile Ile Ile Gly Ile Ile Ile |
| 245 250 255 |
| Tyr Cys Cys Cys Cys Arg Gly Lys Asp Asp Asn Thr Glu Asp Lys Glu |
| 260 265 270 |
| Asp Ala Arg Pro Asn Arg Glu Ala Tyr Glu Glu Pro Pro Glu Gln Leu |
| 275 280 285 |
| Arg Glu Leu Ser Arg Glu Arg Glu Glu Glu Asp Asp Tyr Arg Gln Glu |
| 290 295 300 |
| Glu Gln Arg Ser Thr Gly Arg Glu Ser Pro Asp His Leu Asp Gln |
| 305 310 315 |
It is proposed that the protein contains a 21 amino acid hydrophobic leader sequence which is cleaved to produce a 298 amino acid mature protein with the known N-terminal corrresponding to amino acids 22-319 of SEQ ID NO: 22, i.e.:
| Ile Ser Val Glu Thr Pro Gln Asp Val Leu Arg Ala Ser Gln Gly Lys |
| 5 10 15 |
| Ser Val Thr Leu Pro Cys Thr Tyr His Thr Ser Thr Ser Ser Arg Glu |
| 20 25 30 |
| Gly Leu Ile Gln Trp Asp Lys Leu Leu Leu Thr His Thr Glu Arg Val |
| 35 40 45 |
| Val Ile Trp Pro Phe Ser Asn Lys Asn Tyr Ile His Gly Glu Leu Tyr |
| 50 55 60 |
| Lys Asn Arg Val Ser Ile Ser Asn Asn Ala Glu Gln Ser Asp Ala Ser |
| 65 70 75 80 |
| Ile Thr Ile Asp Gln Leu Thr Met Ala Asp Asn Gly Thr Tyr Glu Cys |
| 85 90 95 |
| Ser Val Ser Leu Met Ser Asp Leu Glu Gly Asn Thr Lys Ser Arg Val |
| 100 105 110 |
| Arg Leu Leu Val Leu Val Pro Pro Ser Lys Pro Glu Cys Gly Ile Glu |
| 115 120 125 |
| Gly Glu Thr Ile Ile Gly Asn Asn Ile Gln Leu Thr Cys Gln Ser Lys |
| 130 135 140 |
| Glu Gly Ser Pro Thr Pro Gln Tyr Ser Trp Lys Arg Tyr Asn Ile Leu |
| 145 150 155 160 |
| Asn Gln Glu Gln Pro Leu Ala Gln Pro Ala Ser Gly Gln Pro Val Ser |
| 165 170 175 |
| Leu Lys Asn Ile Ser Thr Asp Thr Ser Gly Tyr Tyr Ile Cys Thr Ser |
| 180 185 190 |
| Ser Asn Glu Glu Gly Thr Gln Phe Cys Asn Ile Thr Val Ala Val Arg |
| 195 200 205 |
| Ser Pro Ser Met Asn Val Ala Leu Tyr Val Gly Ile Ala Val Gly Val |
| 210 215 220 |
| Val Ala Ala Leu Ile Ile Ile Gly Ile Ile Ile Tyr Cys Cys Cys Cys |
| 225 230 235 240 |
| Arg Gly Lys Asp Asp Asn Thr Glu Asp Lys Glu Asp Ala Arg Pro Asn |
| 245 250 255 |
| Arg Glu Ala Tyr Glu Glu Pro Pro Glu Gln Leu Arg Glu Leu Ser Arg |
| 260 265 270 |
| Glu Arg Glu Glu Glu Asp Asp Tyr Arg Gln Glu Glu Gln Arg Ser Thr |
| 275 280 285 |
| Gly Arg Glu Ser Pro Asp His Leu Asp Gln |
| 290 295 |
The position of the first in-frame stop codon predicts a polypeptide chain, which has a Mr of 33276. Based on a hydrophilicity plot constructed from the amino acid sequence, the molecule appears to have three portions: an extracellular region of 213 amino acids (which by sequence alignment of conserved residues appears to contain two immunoglobulin-like domains), a highly hydrophobic transmembrane domain of 24-27 amino acids, and a highly polar intracellular C-terminal tail. This general structure is suggestive of the molecule being involved in signal transduction.
The cDNA sequence starting at base pair 113 from the 5' end of clone 11, to base pair 1070 of clone 11, which encodes the 298 amino acid protein is as follows (SEQ ID NO: 23):
| ATGGTGGGGA AGATGTGGCC TGTGTTGTGG ACACTCTGTG CAGTCAGGGT GACCGTCGAT |
| GCCATCTCTG TGGAAACTCC GCAGGACGTT CTTCGGGCTT CGCAGGGAAA GAGTGTCACC |
| CTGCCCTGCA CCTACCACAC TTCCACCTCC AGTCGAGAGG GACTTATTCA ATGGGATAAG |
| CTCCTCCTCA CTCATACGGA AAGGGTGGTC ATCTGGCCGT TTTCAAACAA AAACTACATC |
| CATGGTGAGC TTTATAAGAA TCGCGTCAGC ATATCCAACA ATGCTGAGCA GTCCGATGCC |
| TCCATCACCA TTGATCAGCT GACCATGGCT GACAACGGCA CCTACGAGTG TTCTGTCTCG |
| CTGATGTCAG ACCTGGAGGG CAACACCAAG TCACGTGTCC GCCTGTTGGT CCTCGTGCCA |
| CCCTCCAAAC CAGAATGCGG CATCGAGGGA GAGACCATAA TTGGGAACAA CATCCAGCTG |
| ACCTGCCAAT CAAAGGAGGG CTCACCAACC CCTCAGTACA GCTGGAAGAG GTACAACATC |
| CTGAATCAGG AGCAGCCCCT GGCCCAGCCA GCCTCAGGTC AGCCTGTCTC CCTGAAGAAT |
| ATCTCCACAG ACACATCGGG TTACTACATC TGTACCTCCA GCAATGAGGA GGGGACGCAG |
| TTCTGCAACA TCACGGTGGC CGTCAGATCT CCCTCCATGA ACGTGGCCCT GTATGTGGGC |
| ATCGCGGTGG GCGTGGTTGC AGCCCTCATT ATCATTGGCA TCATCATCTA CTGCTGCTGC |
| TGCCGAGGGA AGGACGACAA CACTGAAGAC AAGGAGGATG CAAGGCCGAA CCGGGAAGCC |
| TATGAGGAGC CACCAGAGCA GCTAAGAGAA CTTTCCAGAG AGAGGGAGGA GGAGGATGAC |
| TACAGGCAAG AAGAGCAGAG GAGCACTGGG CGTGAATCCC CGGACCACCT CGACCAGTGA |
Comparison with available DNA and protein databases revealed that the protein consisting of the amino acid sequence sutra (SEQ ID NO: 22) was novel. However, analysis of available expressed sequence tag (EST) databases revealed 74% sequence similarity between part of the human A33 antigen cDNA (nucleotides 286-529) and a 249 base pair EST derived from the murine embryonal carcinoma cell line F9 (EMBL Accession No. MM88A09; DDBJ Accession No. D28657). In the likelihood that this EST corresponded to part of the murine homologue of the human A33 antigen cDNA, sense and antisense PCR primers (17mers) were designed to hybridize to the extremities of the EST clone, as follows:
Primer #1867 (F9 A33 sense) 5'TGACAAAGAAATACATC (SEQ ID NO: 24)
Primer #1868 (F9 A33 antisense) 5'TCTGGCTTGGAGGGTGG (SEQ ID NO: 25)
These primers were used in the touchdowh PCR program described above to amplify a 218 bp product from a normal adult mouse colonic crypt cDNA library. See, e.g. J. Biol. Chem. 268: 27214-27225 (1993) for details on the murine cDNA library used. Briefly the cDNA was reverse transcribed from poly(A)+ enriched RNA purified from adult mouse colon crypt epithelium, and then cloned into the λgt-11 expression vector. This product was gel-purified and DNA sequencing demonstrated that this product closely corresponded to the F9 EST:
| Seq 1 (SEQ ID NO: 26) Seq 2 (SEQ ID NO: 27) |
| MM88A09 EST sequence(nucleic) mouse colon CDNA (PCR |
| product) |
| Seq 1 |
| 5'AGTATCTAACGAGTGCTGAGGTTGTCAAATGCCTCTATCACCATCGACCAGCTGACCATGGA |
| Seq 2 |
| 5'AGTATCTAACGA-TGCTGAG-TTGTCAAATGCCTCTATCACCATCGACCAGCTGACCATGGA |
| Seq 1 |
| CGACAATGGCACCTACGAGTGCTCCGGTGTCACTGATGTCGGACCAGGATGTCAACGCCAA |
| Seq 2 |
| CGACAATGGCACCTACGAGTGCTCCG-TGTCACTGATGTCGGACCAGGATGTCAACGCCAA |
Translation of the murine colonic PCR product revealed significant homology with part of the sequence of the A33 antigen (residues 64-104 of the cleaved molecule, residues 85-125 of SEQ ID NO: 22). The alignment between the predicted human and murine protein sequences is shown below:
Human (amino acids 85-125 of SEQ ID NO: 22)
| 85 |
| Y K N R V S I S N N A E Q S D A S I T I D Q L T M A D N G T Y E C S V |
| S L M S D L |
| 125 |
| Murine (SEQ ID NO: 28) |
| Y E N R V R V S N D A E L S N A S I T I D O L T M D D N G T Y E C S V |
| S L M S D Q |
The F9 PCR product was radiolabelled ([α32 P]ATP and [α32 P] CTP as before) and used as a probe in Northern analysis of multiple murine tissue RNAs (from colonic crypts, small intestinal crypts, kidney, liver, brain, spleen, thymus, lung, pancreas, testis, heart, and thigh muscle). An intense band of approximately 2.6 kb in size was seen only in the lanes containing RNA prepared from colonic crypts and small intestinal crypts, with a very weak signal when testis and pancreas RNA were used. This close correspondence with the size of human A33 antigen mRNA, together with the alignment shown above and the restricted tissue expression, strongly suggested that the F9 clone encodes the murine homologue of the A33 antigen. In addition, these data suggest that the F9 EST contains errors and that the authentic sequence is better described by the sequence of the PCR product described herein.
The F9 PCR product was then used to screen the murine colonic crypt cDNA library described supra, for full length murine A33 cDNA clones. Standard methodologies were used, and twenty clones were identified, most of which contained A33 cDNA inserts of about 2.2 kb; however, two contained longer, 4.2 kb inserts. DNA sequencing was carried out on these two longer clones, and two of the 2.2 kb clones, using standard methods. The 3' sequence of the 4.2 kb clones did not correspond to A33 antigen cDNA, and sequence similarity searching of publicly available libraries using BLAST and FASTA algorithms showed that the 3'-end corresponded to cDNA for stomach, non-muscle Ca2+ ATP-ase.
The 5'-end of all four clones was recognizable as an A33 antigen nucleotide sequence. Hence, the 4.2 kb clone comprises A33 cDNA at its 5'-end, and the stomach non-muscle Ca2+ ATP-ase cDNA at its 3'-end. Sequencing of the 4.2 kb clones showed 2202 base pairs of A33 cDNA. The shorter clones contained 2122 base pairs of cDNA for the antigen. Translation of the longest ORE; predicts a 318 amino acid protein which is not complete at the --NH2 terminus. It shows the same basic structure as the human antigen, and is highly homologous to it. A 20 amino acid hydrophobic leader sequence (missing the initiative methionine) is seen (compared to 21 for human), a V-set, a C2 set immunoglobulin like domain, a 24 amino acid hydrophobic transmembrane domain, and a 61 amino acid intracellular domain are presented. Further, the N-terminal region contains a consensus peptide cleavage site: ADA↓LTVET (SEQ ID NO: 29) which is similar to human cleavage site: ADA↓SVET (SEQ ID NO: 30), each of which produce a mature protein of 298 amino acids.
Overall analysis shows 71% similarity between murine and human sequences in the extracellular domain, 67% in the transmembrane domain, and 54% in the intracellular domain. The murine protein shows 4 potential N-linked glycosylation sites at positions 78, 91, 179 and 202, as compared to the human sequence, which has three potential sites at 91, 179 and 202.
The nucleotide and deduced amino acid sequences for the murine clone are set forth at SEQ ID NOS: 31 and 32. An alignment of the deduced amino acid sequences of human and murine sequences are set forth at FIG. 17.
A further set of experiments were carried out, using the protocols described supra, and the longest A33 antigen cDNA clone (clone 18) was found which also encoded A33. The nucleotide sequence of this clone is presented in SEQ ID NO: 33. This clone is slightly larger than that provided supra for clone 11, in that clone 18 is about 2.8 kilobases long, while clone 11, as indicated, is 2.6 kilobases long. In SEQ ID NO: 29, nucleotides 345 to 1302 appear to encode the amino acid sequence set forth in SEQ ID NO: 33.
As noted, supra, the A33 molecule is believed to be a glycoprotein, with N-linked glycosylation. Additional studies were carried out on relevant post-translational modifications to the antigen.
As reported, supra, the cell lines SW1222, LIM 1215, and COLO 205 are all A33 positive, while SW620 and MF-SH are A33 negative. All of these cell lines were metabolically labelled with 3 H-palmitate, at 500 μCi/ml, then lysed with detergent, and the lysates were precipitated with A33 and FB5, as described supra. FB5, it will be recalled,. serves as a negative control. The precipitates were then subjected to SDS-PAGE analysis, as well as autoradiofluorography.
The results are shown in FIGS. 18 and 19. In FIG. 18, it will be seen that A33 3 H-palmitate labelled A33 antigen, with molecular weights at reduced and non-reduced conditions which were expected. The labelled precipitates were found in all three positive cells, but neither of the negative cells. FB5 was negative in all tests.
When the SDS gels were treated with 1M hydroxylamine (pH 7.5), before autoradiofluorography, staining was abolished, as FIG. 19 shows. This indicates that palmitate groups (acyls), are linked via thioesters. A (CyS)4 domain is found in the molecule, and it is proposed that the palmitate is linked thereto.
The A33 antigen which has now been isolated, characterized and sequenced, can be used to diagnose cancer, colon cancer in particular which is characterized by the expression of the A33 antigen. For example, a sample suspected of containing colon cancer cells is contacted with an antibody specific for the A33 antigen or a fragment thereof, so that A33 protein/antibody complexes can be formed. If these complexes are present, a positive colon cancer diagnosis is indicated.
In addition, the A33 antigen can be used to identify ligands which bind to it (binding partners). The A33 antigen can be isolated, or recombinantly expressed, and used to screen biological sources, including tissue culture media, tissue extracts and cell lysates, for binding partners. Once a binding partner has been found, it is isolated and purified, and can be sequenced. This can be done with the use of a biosensor, in combination with affinity and other chromatographic techniques. Optionally, the A33 antigen can be tagged, to assist in immobilization of the antigen in a specific orientation onto the biosensor surface or affinity support. Identifying binding partners can be done utilizing techniques known to those skilled in the art. See, for example, Stitt et al., Cell, Vol. 80, pp. 661-670 (1995), Nice et al., J. Chromatoaraphy A., Vol. 660, pp. 169-185 (1994) and Bartley et al., Nature, Vol. 368, p. 558 (1994); Lachmann et al., Proc. Natl. Acad. Sci. USA 93: 2523-2527 (1993).
Further, the cDNA encoding A33 antigen has been described herein. This cDNA, including the untranslated portions at the 5' and 3' ends, easily facilitates the production of A33 antigen double-stranded cDNA molecules from tissues and cell lines expressing the A33 antigen, and A33 antigen genomic clones from genomic DNA. To do this, the A33 cDNA is used to design complementary primers for use in the technique of RT-PCR (reverse transcriptase-PCR), a standard procedure for the production of double-stranded cDNA molecules from mRNA templates. Further, the A33 cDNA can be used to design complementary primers for use in standard PCR reactions to amplify portions of the A33 antigen gene from genomic DNA templates.
It is possible that the A33 antigen resides in a novel family of related proteins. The A33 cDNA sequence described herein can be used to design specific and degenerate oligonucleotide primers for use in low stringency PCR reactions to amplify portions of cDNA and genomic DNA molecules encoding proteins related to the A33 antigen. In addition, the A33 cDNA can be used to design specific and degenerate oligonucleotide probes for the identification of members of the A33 antigen gene family by Southern analysis of genomic DNA under low stringency conditions.
These procedures utilizing A33 cDNA are standard procedures, known to those skilled in the art of molecular biology. See, for example, Molecular Cloning: A Laboratory Manual, 2nd edition, 1989 (eds. Sambrook J, Fritsch EF & Maniatis T) Cold Spring Harbor Laboratory Press, U.S.A., and Current Protocols in Molecular Biology Volumes I & II, 1989 (ed. Ausubel, FM) Greene Publishing Associates and Wiley-Interscience, U.S.A.
Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of various aspects of the invention. Thus, it is to be understood that numerous modifications may be made in the illustrative embodiments and other arrangements may be devised without departing from the spirit and scope of the invention. ##STR1## ##STR2##
| NUCLEOTIDE SEQUENCE OF MURINE A33 ANTIGEN |
| CTACCCCTTTGTGAGCAGTCTAGGACTTTGTACACCTGTTAAGTAGGGAGAAGGCAGGGGAGGTGGCTGGTTTAA |
| GGGGA 80 SEQ ID NO: 33 |
| ACTTGAGGGAAGTAGGGAAGACTCCTCTTGGGACCTTTGGAGTAGGTGACACATGAGCCCAGCCCCAGCTCACCT |
| GCCAA 160 |
| TCCAGCTGAGGAGCTCACCTGCCAATCCAGCTGAGGCTGGGCAGAGGTGGGTGAGAAGAGGGAAAATTGCAGGGA |
| CCTCC 240 |
| AGTTGGGCCAGGCCAGAAGCTGCTGTAGCTTTAACCAGACAGCTCAGACCTGTCTGGAGGCTGCCAGTGACAGGT |
| TAGGT 242 |
| TTAGGGCAGAGAAGAAGCAAGACCATGGTGGGGAAGATGTGGCCTGTGTTGTGGACACTCTGTGCAGTCAGGGTG |
| ACCGT 400 |
| CGATGCCATCTCTGTGGAAACTCCGCAGGACGTTCTTCGGGCTTCGCAGGGAAAGAGTGTCACCCTGCCCTGCAC |
| CTACC 480 |
| ACACTTCCACCTCCAGTCGAGAGGGACTTATTCAATGGGATAAGCTCCTCCTCACTCATACGGAAAGGGTGGTCA |
| TCTGG 560 |
| CCGTTTTCAAACAAAAACTACATCCATGGTGAGCTTTATAAGAATCGCGTCAGCATATCCAACAATGCTGAGCAG |
| TCCGA 640 |
| TGCCTCCATCACCATTGATCAGCTGACCATGGCTGACAACGGCACCTACGAGTGTTCTGTCTCGCTGATGTCAGA |
| CCTGG 720 |
| AGGGCAACACCAAGTCACGTGTCCGCCTGTTGGTCCTCGTGCCACCCTCCAAACCAGAATGCGGCATCCAGGGAG |
| AGACC 800 |
| ATAATTGGGAACAACATCCAGCTGACCTGCCAATCAAAGGAGGGCTCACCAACCCCTCAGTACAGCTGGAAGAGG |
| TACAA 880 |
| CATCCTGAATCAGGAGCAGCCCCTGGCCCAGCCAGCCTCAGGTCAGCCTGTCTCCCTGAAGAATATCTCCACAGA |
| CACAT 960 |
| CGGGTTACTACATCTGTACCTCCAGCAATGAGGAGGGGACGCAGTTCTGCAACATCACGGTGGCCGTCAGATCTC |
| CCTCC 1040 |
| ATGAACGTGGCCCTGTATGTGCGCATCGCGGTGGGCGTGGTTGCAGCCCTCATTATCATTGGCATCATCATCTAC |
| TGCTG 1120 |
| CTGCTGCCGAGGGAAGGACGACAACACTGAAGACAAGGACGATGCAAGGCCGAACCGGGAAGCCTATGAGGAGCC |
| ACCAG 1200 |
| AGCAGCTAAGAGAACTTTCCAGAGAGAGGGAGGAGGAGGATGACTACAGGCAAGAAGAGCAGAGGAGCACTGGGC |
| GTGAA 1280 |
| TCCCCGGACCACCTCGACCAGTGACAGGCCAGCAGCAGAGGGCGGCGGAGGAAGGGTTAGGGGTTCATTCTCCCG |
| CTTCC 1320 |
| TGGCCTCCCTTCTCCTTTCTAAGCCCTGTTCTCCTGTCCCTCCATCCCAGACATTGATGGGGACATTTCTTCCCC |
| AGTGT 1440 |
| CAGCTGTGGGGAACATGGCTGGCCTGGTAAGGGGGTCCCTGTGCTGATCCTGCTGACCTCACTGTCCTGTGAAGT |
| AACCC 1520 |
| CTCCTGGCTGTGACACCTGGTGCGGGCCTGGCCCTCACTCAAGACCAGGCTGCAGCCTCCACTTCCCTCGTAGTT |
| GGCAG 1600 |
| GAGCTCCTGGAAGCACAGCGCTGAGCATGGGGCGCTCCCACTCAGAACTCTCCAGGGAGGCGATGCCAGCCTTGG |
| GGGGT 1680 |
| GGGGGCTGTCCTGCTCACCTGTGTGCCCAGCACCTGGAGGGGCACCAGGTGGAGGGTTTGCACTCCACACATCTT |
| TCTTG 1760 |
| AATGAATGAAAGAATAAGTGAGTATGCTTGGCCCCTGCATTGGCCTGGCCTCCAGCTCCCACTCCCTTTCCAACC |
| TCACT 1840 |
| TCCCGTAGCTGCCAGTATGTTCCAAACCCTCCTGGGAAGGCCACCTCCCACTCCTGCTGCACAGGCCCTGGGGAG |
| CTTTT 1920 |
| GCCCACACACTTTCCATCTCTGCCTGTCAATATCGTACCTGTCCCTCCAGGCCCATCTCAAATCACAAGGATTTC |
| TCTAA 2000 |
| CCCTATCCTAATTGTCCACATACGTGGAAACAATCCTGTTACTCTGTCCCACGTGGAATCATGGGCCACAAGGCA |
| CAGTC 2090 |
| TTCTGAGCGAGTGCTCTCACTGTATTAGAGCGCCAGCTCCTTGGGGCAGGGCCTGGGCCTCATGGCTTTTGCTTT |
| CCCTG 2160 |
| AAGCCCTAGTAGCTGGCGCCCATCCTAGTGGGCACTTAAGCTTAATTGGGGAAACTGCTTTGATTGGTTGTGCCT |
| TCCCT 2240 |
| TCTCTGGTCTCCTTGAGATGATCGTAGACACAGGGATGATTCCCACCCAAACCCACGTATTCATTCAGTGAGTTA |
| AACAC 2320 |
| GAATTGATTTAAAGTGAACACACACAAGGGAGCTTGCTTGCAGATGGTCTGAGTTCTTGTGTCCTGGTAATTCCT |
| CTCCA 2400 |
| GGCCAGAATAATTGGCATGTCTCCTCAACCCACATGGGGTTCCTGGTTGTTCCTGCATCCCGATACCTCAGCCCT |
| CGCCC 2480 |
| TGCCCAGCCCATTTGGGCTCTGGTTTTCTGGTGGGGCTGTCCTGCTGCCCTCCCACAGCCTCCTTCTGTTTGTCG |
| AGCAT 2520 |
| TTCTTCTACTCTTGAGAGCTCAGGCAGCGTTAGGGCTGCTTAGGTCTCATGGACCACTGGCTGGTCTCACCCAAC |
| TGCAG 2640 |
| TTTACTATTGCTATCTTTTCTGGATGATCAGAAAAATAATTCCATAAATCTATTGTCTACTTGCGATTTTTTAAA |
| AAATG 2720 |
| TATATTTTTATATATATTGTTAAATCCTTTGCTTCATTCCAAATGCTTTCAGTAATAATAAAATTGTGGGTGGAA |
| AAAAA 2800 |
| AAA |
| 2803 |
| SEQUENCE LISTING |
| (1) GENERAL INFORMATION: |
| (iii) NUMBER OF SEQUENCES: 33 |
| (2) INFORMATION FOR SEQ ID NO: 1: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 38 amino acids |
| (B) TYPE: amino acid |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 |
| Xaa Ser Val Glu Thr Pro Gln Asp Val Leu Arg Ala Ser Gln Gly Lys |
| 5 10 15 |
| Ser Val Thr Leu Pro Xaa Thr Tyr His Thr Ser Xaa Xaa Xaa Arg Glu |
| 20 25 30 |
| Gly Leu Ile Gln Trp Asp |
| 35 |
| (2) INFORMATION FOR SEQ ID NO: 2: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 11 amino acids |
| (B) TYPE: amino acid |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 |
| Glu Ala Tyr Glu Glu Pro Pro Glu Gln Leu Arg |
| 5 10 |
| (2) INFORMATION FOR SEQ ID NO: 3: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 9 amino acids |
| (B) TYPE: amino acid |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 |
| Val Val Ile Trp Pro Phe Ser Asn Lys |
| 5 |
| (2) INFORMATION FOR SEQ ID NO: 4: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 40 amino acids |
| (B) TYPE: amino acid |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 |
| Ile Ser Val Glu Thr Pro Gln Asp Val Leu Arg Ala Ser Gln Gly Lys |
| 5 10 15 |
| Ser Val Thr Leu Pro Xaa Thr Tyr His Thr Ser Thr Ser Ser Arg Glu |
| 20 25 30 |
| Gly Leu Ile Gln Trp Asp Lys Leu |
| 35 40 |
| (2) INFORMATION FOR SEQ ID NO: 5: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 30 amino acids |
| (B) TYPE: amino acid |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5 |
| Asp Val Leu Arg Ala Ser Gln Gly Lys Ser Val Thr Leu Pro Cys Thr |
| 5 10 15 |
| Tyr His Thr Ser Thr Ser Ser Arg Glu Gly Leu Ile Gln Trp |
| 20 25 30 |
| (2) INFORMATION FOR SEQ ID NO: 6: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 39 amino acids |
| (B) TYPE: amino acid |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6 |
| Asp Lys Leu Leu Leu Thr His Thr Glu Arg Val Val Ile Trp Pro Phe |
| 5 10 15 |
| Ser Asn Lys Asn Tyr Ile His Gly Glu Leu Tyr Lys Asn Arg Val Ser |
| 20 25 30 |
| Ile Ser Asn Asn Ala Glu Gln |
| 35 |
| (2) INFORMATION FOR SEQ ID NO: 7: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 15 amino acids |
| (B) TYPE: amino acid |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7 |
| Glu Leu Tyr Lys Asn Arg Val Ser Ile Ser Asn Asn Ala Glu Gln |
| 5 10 15 |
| (2) INFORMATION FOR SEQ ID NO: 8: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 12 amino acids |
| (B) TYPE: amino acid |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8 |
| Asp Xaa Gly Thr Tyr Glu Cys Ser Val Ser Leu Met |
| 5 10 |
| (2) INFORMATION FOR SEQ ID NO: 9: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 16 amino acids |
| (B) TYPE: amino acid |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 |
| Ile Gln Leu Thr Cys Gln Ser Lys Glu Gly Ser Pro Thr Pro Gln Tyr |
| 5 10 15 |
| (2) INFORMATION FOR SEQ ID NO: 10: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 21 amino acids |
| (B) TYPE: amino acid |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10 |
| Leu Val Leu Val Pro Pro Ser Lys Pro Glu Cys Gly Ile Glu Gly Glu |
| 5 10 15 |
| Thr Ile Ile Gly Asn |
| 20 |
| (2) INFORMATION FOR SEQ ID NO: 11: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 17 amino acids |
| (B) TYPE: amino acid |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11 |
| Ile Leu Asn Gln Glu Gln Pro Leu Ala Gln Pro Ala Ser Gly Gln Pro |
| 5 10 15 |
| Val |
| (2) INFORMATION FOR SEQ ID NO: 12: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 17 nucleotides |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12 |
| ARYTTRTCCC ACTGAAT 17 |
| (2) INFORMATION FOR SEQ ID NO: 13: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 17 nucleotides |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13 |
| ARYTTRTCCC ATTGAAT 17 |
| (2) INFORMATION FOR SEQ ID NO: 14: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 17 nucleotides |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14 |
| ARYTTRTCCC ACTGGAT 17 |
| (2) INFORMATION FOR SEQ ID NO: 15: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 17 nucleotides |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15 |
| ARYTTRTCCC ATTGGAT 17 |
| (2) INFORMATION FOR SEQ ID NO: 16: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 17 nucleotides |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16 |
| ARYTTRTCCC ACTGTAT 17 |
| (2) INFORMATION FOR SEQ ID NO: 17: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 17 nucleotides |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17 |
| ARYTTRTCCC ATTGTAT 17 |
| (2) INFORMATION FOR SEQ ID NO: 18: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 17 nucleotides |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18 |
| CGAGGTCGAC GGTATCG 17 |
| (2) INFORMATION FOR SEQ ID NO: 19: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 20 nucleotides |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19 |
| CCTGTCTGGA GGCTGCCAGT 20 |
| (2) INFORMATION FOR SEQ ID NO: 20: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 20 nucleotides |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20 |
| AGGTGCAGGG CAGGGTGACA 20 |
| (2) INFORMATION FOR SEQ ID NO: 21: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 11 nucleotides |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21 |
| GCCCRCCATG G 11 |
| (2) INFORMATION FOR SEQ ID NO: 22: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 319 amino acids |
| (B) TYPE: amino acid |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22 |
| Met Val Gly Lys Met Trp Pro Val Leu Trp Thr Leu Cys Ala Val Arg |
| 5 10 15 |
| Val Thr Val Asp Ala Ile Ser Val Glu Thr Pro Gln Asp Val Leu Arg |
| 20 25 30 |
| Ala Ser Gln Gly Lys Ser Val Thr Leu Pro Cys Thr Tyr His Thr Ser |
| 35 40 45 |
| Thr Ser Ser Arg Glu Gly Leu Ile Gln Trp Asp Lys Leu Leu Leu Thr |
| 50 55 60 |
| His Thr Glu Arg Val Val Ile Trp Pro Phe Ser Asn Lys Asn Tyr Ile |
| 65 70 75 80 |
| His Gly Glu Leu Tyr Lys Asn Arg Val Ser Ile Ser Asn Asn Ala Glu |
| 85 90 95 |
| Gln Ser Asp Ala Ser Ile Thr Ile Asp Gln Leu Thr Met Ala Asp Asn |
| 100 105 110 |
| Gly Thr Tyr Glu Cys Ser Val Ser Leu Met Ser Asp Leu Glu Gly Asn |
| 115 120 125 |
| Thr Lys Ser Arg Val Arg Leu Leu Val Leu Val Pro Pro Ser Lys Pro |
| 130 135 140 |
| Glu Cys Gly Ile Glu Gly Glu Thr Ile Ile Gly Asn Asn Ile Gln Leu |
| 145 150 155 160 |
| Thr Cys Gln Ser Lys Glu Gly Ser Pro Thr Pro Gln Tyr Ser Trp Lys |
| 165 170 175 |
| Arg Tyr Asn Ile Leu Asn Gln Glu Gln Pro Leu Ala Gln Pro Ala Ser |
| 180 185 190 |
| Gly Gln Pro Val Ser Leu Lys Asn Ile Ser Thr Asp Thr Ser Gly Tyr |
| 195 200 205 |
| Tyr Ile Cys Thr Ser Ser Asn Glu Glu Gly Thr Gln Phe Cys Asn Ile |
| 210 215 220 |
| Thr Val Ala Val Arg Ser Pro Ser Met Asn Val Ala Leu Tyr Val Gly |
| 225 230 235 240 |
| Ile Ala Val Gly Val Val Ala Ala Leu Ile Ile Ile Gly Ile Ile Ile |
| 245 250 255 |
| Tyr Cys Cys Cys Cys Arg Gly Lys Asp Asp Asn Thr Glu Asp Lys Glu |
| 260 265 270 |
| Asp Ala Arg Pro Asn Arg Glu Ala Tyr Glu Glu Pro Pro Glu Gln Leu |
| 275 280 285 |
| Arg Glu Leu Ser Arg Glu Arg Glu Glu Glu Asp Asp Tyr Arg Gln Glu |
| 290 295 300 |
| Glu Gln Arg Ser Thr Gly Arg Glu Ser Pro Asp His Leu Asp Gln |
| 305 310 315 |
| (2) INFORMATION FOR SEQ ID NO: 23: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 960 nucleotides |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23 |
| ATGGTGGGGA AGATGTGGCC TGTGTTGTGG ACACTCTGTG CAGTCAGGGT GACCGTCGAT 60 |
| GCCATCTCTG TGGAAACTCC GCAGGACGTT CTTCGGGCTT CGCAGGGAAA GAGTGTCACC 120 |
| CTGCCCTGCA CCTACCACAC TTCCACCTCC AGTCGAGAGG GACTTATTCA ATGGGATAAG 180 |
| CTCCTCCTCA CTCATACGGA AAGGGTGGTC ATCTGGCCGT TTTCAAACAA AAACTACATC 240 |
| CATGGTGAGC TTTATAAGAA TCGCGTCAGC ATATCCAACA ATGCTGAGCA GTCCGATGCC 300 |
| TCCATCACCA TTGATCAGCT GACCATGGCT GACAACGGCA CCTACGAGTG TTCTGTCTCG 360 |
| CTGATGTCAG ACCTGGAGGG CAACACCAAG TCACGTGTCC GCCTGTTGGT CCTCGTGCCA 420 |
| CCCTCCAAAC CAGAATGCGG CATCGAGGGA GAGACCATAA TTGGGAACAA CATCCAGCTG 480 |
| ACCTGCCAAT CAAAGGAGGG CTCACCAACC CCTCAGTACA GCTGGAAGAG GTACAACATC 540 |
| CTGAATCAGG AGCAGCCCCT GGCCCAGCCA GCCTCAGGTC AGCCTGTCTC CCTGAAGAAT 600 |
| ATCTCCACAG ACACATCGGG TTACTACATC TGTACCTCCA GCAATGAGGA GGGGACGCAG 660 |
| TTCTGCAACA TCACGGTGGC CGTCAGATCT CCCTCCATGA ACGTGGCCCT GTATGTGGGC 720 |
| ATCGCGGTGG GCGTGGTTGC AGCCCTCATT ATCATTGGCA TCATCATCTA CTGCTGCTGC 780 |
| TGCCGAGGGA AGGACGACAA CACTGAAGAC AAGGAGGATG CAAGGCCGAA CCGGGAAGCC 840 |
| TATGAGGAGC CACCAGAGCA GCTAAGAGAA CTTTCCAGAG AGAGGGAGGA GGAGGATGAC 900 |
| TACAGGCAAG AAGAGCAGAG GAGCACTGGG CGTGAATCCC CGGACCACCT CGACCAGTGA 960 |
| (2) INFORMATION FOR SEQ ID NO: 24: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 17 nucleotides |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24 |
| TGACAAAGAA ATACATC 17 |
| (2) INFORMATION FOR SEQ ID NO: 25: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 17 nucleotides |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25 |
| TCTGGCTTGG AGGGTGG 17 |
| (2) INFORMATION FOR SEQ ID NO: 26: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 123 nucleotides |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26 |
| AGTATCTAAC GAGTGCTGAG GTTGTCAAAT GCTGACCATG GACGACAATG GCACCTACGA 60 |
| GTGCTCCGGT GTCACTGATG GCCTCTATCA CCATCGACCA TCGGACCAGG ATGTCAACGC 120 |
| CAA 123 |
| (2) INFORMATION FOR SEQ ID NO: 27: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 120 nucleotides |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27 |
| AGTATCTAAC GATGCTGAGT TGTCAAATGC CTCTATCAGC TGACCATGGA CGACAATGGC 60 |
| ACCTACGAGT GCTCCGTGTC ACTGATGCCA TCGACCATCG GACCAGGATG TCAACGCCAA 120 |
| (2) INFORMATION FOR SEQ ID NO: 28: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 41 amino acids |
| (B) TYPE: amino acid |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28 |
| Tyr Glu Asn Arg Val Arg Val Ser Asn Asp Ala Glu Lys Ser Asn |
| 5 10 15 |
| Ala Ser Ile Thr Ile Asp Gln Lys Thr Met Asp Asp Asn Gly Thr |
| 20 25 30 |
| Tyr Glu Cys Ser Val Ser Leu Met Ser Asp Gln |
| 35 40 |
| (2) INFORMATION FOR SEQ ID NO: 29: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 8 amino acids |
| (B) TYPE: amino acid |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29 |
| Ala Asp Ala Leu Thr Val Glu Thr |
| 5 |
| (2) INFORMATION FOR SEQ ID NO: 30: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 8 amino acids |
| (B) TYPE: amino acid |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30 |
| Ala Asp Ala Ile Ser Val Glu Thr |
| 5 |
| (2) INFORMATION FOR SEQ ID NO: 31: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 2565 nucleotides |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31 |
| GGGACTCCAG TTGGGCCAGG CCAGAAGCTG CTGTAGCTTT AACCAGACAG CTCAGACCTG 60 |
| TCTGGAGGCT GCCAGTGACA GGTTAGGTTT AGGGCAGAGA AGAAGCAAGA CCATGGTGGG 120 |
| GAAGATGTGG CCTGTGTTGT GGACACTCTG TGCAGTCAGG GTGACCGTCG ATGCCATCTC 180 |
| TGTGGAAACT CCGCAGGACG TTCTTCGGGC TTCGCAGGGA AAGAGTGTCA CCCTGCCCTG 240 |
| CACCTACCAC ACTTCCACCT CCAGTCGAGA GGGACTTATT CAATGGGATA AGCTCCTCCT 300 |
| CACTCATACG GAAAGGGTGG TCATCTGGCC GTTTTCAAAC AAAAACTACA TCCATGGTGA 360 |
| GCTTTATAAG AATCGCGTCA GCATATCCAA CAATGCTGAG CAGTCCGATG CCTCCATCAC 420 |
| CATTGATCAG CTGACCATGG CTGACAACGG CACCTACGAG TGTTCTGTCT CGCTGATGTC 480 |
| AGACCTGGAG GGCAACACCA AGTCACGTGT CCGCCTGTTG GTCCTCGTGC CACCCTCCAA 540 |
| ACCAGAATGC GGCATCGAGG GAGAGACCAT AATTGGGAAC AACATCCAGC TGACCTGCCA 600 |
| ATCAAAGGAG GGCTCACCAA CCCCTCAGTA CAGCTGGAAG AGGTACAACA TCCTGAATCA 660 |
| GGAGCAGCCC CTGGCCCAGC CAGCCTCAGG TCAGCCTGTC TCCCTGAAGA ATATCTCCAC 720 |
| AGACACATCG GGTTACTACA TCTGTACCTC CAGCAATGAG GAGGGGACGC AGTTCTGCAA 780 |
| CATCACGGTG GCCGTCAGAT CTCCCTCCAT GAACGTGGCC CTGTATGTGG GCATCGCGGT 840 |
| GGGCGTGGTT GCAGCCCTCA TTATCATTGG CATCATCATC TACTGCTGCT GCTGCCGAGG 900 |
| GAAGGACGAC AACACTGAAG ACAAGGAGGA TGCAAGGCCG AACCGGGAAG CCTATGAGGA 960 |
| GCCACCAGAG CAGCTAAGAG AACTTTCCAG AGAGAGGGAG GAGGAGGATG ACTACAGGCA 1020 |
| AGAAGAGCAG AGGAGCACTG GGCGTGAATC CCCGGACCAC CTCGACCAGT GACAGGCCAG 1080 |
| CAGCAGAGGG CGGCGGAGGA AGGGTTAGGG GTTCATTCTC CCGCTTCCTG GCCTCCCTTC 1140 |
| TCCTTTCTAA GCCCTGTTCT CCTGTCCCTC CATCCCAGAC ATTGATGGGG ACATTTCTTC 1200 |
| CCCAGTGTCA GCTGTGGGGA ACATGGCTGG CCTGGTAAGG GGGTCCCTGT GCTGATCCTG 1260 |
| CTGACCTCAC TGTCCTGTGA AGTAACCCCT CCTGGCTGTG ACACCTGGTG CGGGCCTGCC 1320 |
| CTCACTCAAG ACCAGGCTGC AGCCTCCACT TCCCTCGTAG TTGGCAGGAG CTCCTGGAGA 1380 |
| GCACAGCGCT GAGCATGGGG CGCTCCCACT CAGAACTCTC CAGGGAGGCG ATGCCAGCCT 1440 |
| TGGGGGGTGG GGGCTGTCCT GCTCACCTGT GTGCCCAGCA CCTGGAGGGG CACCAGGTGG 1500 |
| AGGGTTTGCA CTCCACACAT CTTTCTTGAA TGAATGAAAG AATAAGTGAG TATGCTTGGG 1560 |
| CCCTGCATTG GCCTGGCCTC CAGCTCCCAC TCCCTTTCCA ACCTCACTTC CCGTAGCTGC 1620 |
| CAGTATGTTC CAAACCCTCC TGGGAAGGCC ACCTCCCACT CCTGCTGCAC AGGCCCTGGG 1680 |
| GAGCTTTTGC CCACACACTT TCCATCTCTG CCTGTCAATA TCGTACCTGT CCCTCCAGGC 1740 |
| CCATCTCAAA TCACAAGGAT TTCTCTAACC CTATCCTAAT TGTCCACATA CGTGGAAACA 1800 |
| ATCCTGTTAC TCTGTCCCAC GTCCAATCAT GGGCCACAAG GCACAGTCTT CTGAGCGAGT 1860 |
| GCTCTCACTG TATTAGAGCG CCAGCTCCTT GGGGCAGGGC CTGGGCCTCA TGGCTTTTGC 1920 |
| TTTCCCTGAA GCCCTAGTAG CTGGCGCCCA TCCTAGTGGG CACTTAAGCT TAATTGGGGA 1980 |
| AACTGCTTTG ATTGGTTGTG CCTTCCCTTC TCTGGTCTCC TTGAGATGAT CGTAGACACA 2040 |
| GGGATGATTC CCACCCAAAC CCACGTATTC ATTCAGTGAG TTAAACACGA ATTGATTTAA 2100 |
| AGTGAACACA CACAAGGGAG CTTGCTTGCA GATGGTCTGA GTTCTTGTGT CCTGGTAATT 2160 |
| CCTCTCCAGG CCAGAATAAT TGGCATGTCT CCTCAACCCA CATGGGGTTC CTGGTTGTTC 2220 |
| CTGCATCCCG ATACCTCAGC CCTGGCCCTG CCCAGCCCAT TTGGGCTCTG GTTTTCTGGT 2280 |
| GGGNCTGTCC TGCTGCCCTC CCACNAGCCT CCTTCTGTTT GTCGAGCATT TCTTCTACTC 2340 |
| TTNAGAGCTC AGGCAGCGTT AGGGCTGCTT AGGTCTCATG GACCAGTGGC TGGTCTCACC 2400 |
| CAACTGCAGT TTACTATTGC TATCTTTTCT GGATGATCAG AAAAATAATT CCATAAATCT 2460 |
| ATTGTCTACT TGCGATTTTT TAAAAAATGT ATATTTTTAT ATATATTGTT AAATCCTTTG 2520 |
| CTTCATTCCA AATGCTTTCA GTAATAATAA AATTGTGGGT GGAAA 2565 |
| (2) INFORMATION FOR SEQ ID NO: 32: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 318 amino acids |
| (B) TYPE: amino acid |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32 |
| Leu Gly Lys Ala Gly Ser Val Val Trp Met Leu Cys Ala Ile Trp Val |
| 5 10 15 |
| Ala Ala Asp Ala Leu Thr Val Glu Thr Thr Gln Asp Ile Leu Arg Ala |
| 20 25 30 |
| Ala Arg Gly Arg Ser Val Thr Leu Pro Cys Thr Tyr Asn Thr Tyr Val |
| 35 40 45 |
| Ser Asp Arg Glu Gly Phe Ile Gln Trp Asp Lys Leu Leu Arg Ser Gln |
| 50 55 60 |
| Thr Glu Arg Val Val Thr Trp Asn Phe Val Thr Lys Lys Tyr Ile Tyr |
| 65 70 75 80 |
| Gly Asn Arg Tyr Glu Asn Arg Val Arg Val Ser Asn Asp Ala Glu Leu |
| 85 90 95 |
| Ser Asn Ala Ser Ile Thr Ile Asp Gln Leu Thr Met Asp Asp Asn Gly |
| 100 105 110 |
| Thr Tyr Glu Cys Ser Val Ser Leu Met Ser Asp Gln Asp Val Asn Ala |
| 115 120 125 |
| Lys Ser Arg Val Arg Leu Leu Val Leu Val Pro Pro Ser Lys Pro Asp |
| 130 135 140 |
| Cys Ser Ile Gln Gly Glu Met Val Ile Gly Asn Asn Ile Gln Leu Thr |
| 145 150 155 160 |
| Cys His Ser Ala Glu Gly Ser Pro Ser Pro Gln Tyr Ser Trp Lys Ser |
| 165 170 175 |
| Tyr Asn Ala Gln Asn Gln Gln Arg Pro Leu Thr Gln Pro Val Ser Gly |
| 180 185 190 |
| Glu Pro Leu Leu Leu Lys Asn Ile Ser Thr Glu Thr Ala Gly Tyr Tyr |
| 195 200 205 |
| Ile Cys Thr Ser Ser Asn Asp Val Gly Ile Glu Ser Cys Asn Ile Thr |
| 210 215 220 |
| Val Ala Pro Arg Pro Pro Ser Met Asn Ile Ala Leu Tyr Ala Gly Ile |
| 225 230 235 240 |
| Ala Gly Ser Val Phe Val Ala Leu Ile Ile Ile Gly Val Ile Val Tyr |
| 245 250 255 |
| Cys Cys Cys Cys Arg Glu Lys Asp Asp Lys Asp Gln Asp Arg Glu Asp |
| 260 265 270 |
| Ala Arg Pro Asn Arg Ala Ala Tyr Gln Val Pro Lys Lys Glu Gln Lys |
| 275 280 285 |
| Glu Ile Ser Arg Gly Arg Glu Asp Glu Asp Asp His Arg His Glu Asp |
| 290 295 300 |
| Arg Trp Ser Ser Gly Arg Ser Thr Pro Asp Gln Pro Phe Gln |
| 305 310 315 |
| (2) INFORMATION FOR SEQ ID NO: 33: |
| (i) SEQUENCE CHARACTERISTICS: |
| (A) LENGTH: 2803 nucleotides |
| (B) TYPE: nucleic acid |
| (C) STRANDEDNESS: single |
| (D) TOPOLOGY: linear |
| (vi) ORIGINAL SOURCE: |
| (A) ORGANISM: not provided |
| (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33 |
| CTACCCCTTT GTGAGCAGTC TAGGACTTTG TACACCTGTT AAGTAGGGAG 50 |
| AAGGCAGGGG AGGTGGCTGG TTTAAGGGGA ACTTGAGGGA AGTAGGGAAG 100 |
| ACTCCTCTTG GGACCTTTGG AGTAGGTGAC ACATGAGCCC AGCCCCAGCT 150 |
| CACCTGCCAA TCCAGCTGAG GAGCTCACCT GCCAATCCAG CTGAGGCTGG 200 |
| GCAGAGGTGG GTGAGAAGAG GGAAAATTGC AGGGACCTCC AGTTGGGCCA 250 |
| GGCCAGAAGC TGCTGTAGCT TTAACCAGAC AGCTCAGACC TGTCTGGAGG 300 |
| CTGCCAGTGA CAGGTTAGGT TTAGGGCAGA GAAGAAGCAA GACCATGGTG 350 |
| GGGAAGATGT GGCCTGTGTT GTGGACACTC TGTGCAGTCA GGGTGACCGT 400 |
| CGATGCCATC TCTGTGGAAA CTCCGCAGGA CGTTCTTCGG GCTTCGCAGG 450 |
| GAAAGAGTGT CACCCTGCCC TGCACCTACC ACACTTCCAC CTCCAGTCGA 500 |
| GAGGGACTTA TTCAATGGGA TAAGCTCCTC CTCACTCATA CGGAAAGGGT 550 |
| GGTCATCTGG CCGTTTTCAA ACAAAAACTA CATCCATGGT GAGCTTTATA 600 |
| AGAATCGCGT CAGCATATCC AACAATGCTG AGCAGTCCGA TGCCTCCATC 650 |
| ACCATTGATC AGCTGACCAT GGCTGACAAC GGCACCTACG AGTGTTCTGT 700 |
| CTCGCTGATG TCAGACCTGG AGGGCAACAC CAAGTCACGT GTCCGCCTGT 750 |
| TGGTCCTCGT GCCACCCTCC AAACCAGAAT GCGGCATCGA GGGAGAGACC 800 |
| ATAATTGGGA ACAACATCCA GCTGACCTGC CAATCAAAGG AGGGCTCACC 850 |
| AACCCCTCAG TACAGCTGGA AGAGGTACAA CATCCTGAAT CAGGAGCAGC 900 |
| CCCTGGCCCA GCCAGCCTCA GGTCAGCCTG TCTCCCTGAA GAATATCTCC 950 |
| ACAGACACAT CGGGTTACTA CATCTGTACC TCCAGCAATG AGGAGGGGAC 1000 |
| GCAGTTCTGC AACATCACGG TGGCCGTCAG ATCTCCCTCC ATGAACGTGG 1050 |
| CCCTGTATGT GGGCATCGCG GTGGGCGTGG TTGCAGCCCT CATTATCATT 1100 |
| GGCATCATCA TCTACTGCTG CTGCTGCCGA GGGAAGGACG ACAACACTGA 1150 |
| AGACAAGGAG GATGCAAGGC CGAACCGGGA AGCCTATGAG GAGCCACCAG 1200 |
| AGCAGCTAAG AGAACTTTCC AGAGAGAGGG AGGAGGAGGA TGACTACAGG 1250 |
| CAAGAAGAGC AGAGGAGCAC TGGGCGTGAA TCCCCGGACC ACCTCGACCA 1300 |
| GTGACAGGCC AGCAGCAGAG GGCGGCGGAG GAAGGGTTAG GGGTTCATTC 1350 |
| TCCCGCTTCC TGGCCTCCCT TCTCCTTTCT AAGCCCTGTT CTCCTGTCCC 1400 |
| TCCATCCCAG ACATTGATGG GGACATTTCT TCCCCAGTGT CAGCTGTGGG 1450 |
| GAACATGGCT GGCCTGGTAA GGGGGTCCCT GTGCTGATCC TGCTGACCTC 1500 |
| ACTGTCCTGT GAAGTAACCC CTCCTGGCTG TGACACCTGG TGCGGGCCTG 1550 |
| GCCCTCACTC AAGACCAGGC TGCAGCCTCC ACTTCCCTCG TAGTTGGCAG 1600 |
| GAGCTCCTGG AAGCACAGCG CTGAGCATGG GGCGCTCCCA CTCAGAACTC 1650 |
| TCCAGGGAGG CGATGCCAGC CTTGGGGGGT GGGGGCTGTC CTGCTCACCT 1700 |
| GTGTGCCCAG CACCTGGAGG GGCACCAGGT GGAGGGTTTG CACTCCACAC 1750 |
| ATCTTTCTTG AATGAATGAA AGAATAAGTG AGTATGCTTG GGCCCTGCAT 1800 |
| TGGCCTGGCC TCCAGCTCCC ACTCCCTTTC CAACCTCACT TCCCGTAGCT 1850 |
| GCCAGTATGT TCCAAACCCT CCTGGGAAGG CCACCTCCCA CTCCTGCTGC 1900 |
| ACAGGCCCTG GGGAGCTTTT GCCCACACAC TTTCCATCTC TGCCTGTCAA 1950 |
| TATCGTACCT GTCCCTCCAG GCCCATCTCA AATCACAAGG ATTTCTCTAA 2000 |
| CCCTATCCTA ATTGTCCACA TACGTGGAAA CAATCCTGTT ACTCTGTCCC 2050 |
| ACGTCCAATC ATGGGCCACA AGGCACAGTC TTCTGAGCGA GTGCTCTCAC 2100 |
| TGTATTAGAG CGCCAGCTCC TTGGGGCAGG GCCTGGGCCT CATGGCTTTT 2150 |
| GCTTTCCCTG AAGCCCTAGT AGCTGGCGCC CATCCTAGTG GGCACTTAAG 2200 |
| CTTAATTGGG GAAACTGCTT TGATTGGTTG TGCCTTCCCT TCTCTGGTCT 2250 |
| CCTTGAGATG ATCGTAGACA CAGGGATGAT TCCCACCCAA ACCCACGTAT 2300 |
| TCATTCAGTG AGTTAAACAC GAATTGATTT AAAGTGAACA CACACAAGGG 2350 |
| AGCTTGCTTG CAGATGGTCT GAGTTCTTGT GTCCTGGTAA TTCCTCTCCA 2400 |
| GGCCAGAATA ATTGGCATGT CTCCTCAACC CACATGGGGT TCCTGGTTGT 2450 |
| TCCTGCATCC CGATACCTCA GCCCTGGCCC TGCCCAGCCC ATTTGGGCTC 2500 |
| TGGTTTTCTG GTGGGGCTGT CCTGCTGCCC TCCCACAGCC TCCTTCTGTT 2550 |
| TGTCGAGCAT TTCTTCTACT CTTGAGAGCT CAGGCAGCGT TAGGGCTGCT 2600 |
| TAGGTCTCAT GGACCAGTGG CTGGTCTCAC CCAACTGCAG TTTACTATTG 2650 |
| CTATCTTTTC TGGATGATCA GAAAAATAAT TCCATAAATC TATTGTCTAC 2700 |
| TTGCGATTTT TTAAAAAATG TATATTTTTA TATATATTGT TAAATCCTTT 2750 |
| GCTTCATTCC AAATGCTTTC AGTAATAATA AAATTGTGGG TGGAAAAAAA 2800 |
| AAA 2803 |
Ji, Hong, Ritter, Gerd, Welt, Sydney, Burgess, Antony, White, Sara, Johnstone, Cameron, Old, Lloyd J, Catimel, Bruno, Heath, Joan, Simpson, Richard J, Nice, Edouard, Moritz, R. L
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